Tube formation is a nearly ubiquitous process required in metazoans to construct the thoroughfares through which the various liquids and gases that sustain life flow. We focus on tube formation in the model system of the Drosophila embryonic trachea, a genetically tractable model for learning the molecules and mechanisms underlying the organization of cells into tubular organs. This application focuses on events downstream of a key transcription factor gene required for normal tube formation: trachealess (trh), which encodes a bHLH-PAS transcription factor. Trh functions as a major regulator of tube formation, not only in the trachea but also in two other tube-forming organs. In trh mutants, the primordia for these tissues remain on the embryo surface at their site of origin, failing to undergo any of the movements of tube morphogenesis. With the recent advances we have made in live imaging of tracheal morphogenesis, the identification of several early expressed Trh target genes and the discovery that two of the Trh target genes play novel roles in tube size control, we are thus poised to carry our analysis to the next level; linking molecular changes in target genes to the cell biological events of tube morphogenesis. In this proposal, we have three specific aims: (1) We unravel the roles of new Trh target genes that are expressed early and encode molecules with domain structures that suggest a role in mediating tube invagination. (2) We explore the mechanisms whereby Sano and the PCP regulators control tube size through effects on cell shape. (3) We link the molecular activity of an unusual enzyme encoded by mipp1 to tube size control and migration. For these studies, we use the collective experience of the lab to take advantage of all the tools Drosophila has to offer, including a well annotated genome, methodologies for cleanly eliminating gene function either in the whole animal or in a subset of cells, mechanisms to express both untagged and tagged versions of a given protein in any tissue, as well as methods for imaging both fixed and living tissues. We expect these studies to implicate new molecules and reveal novel mechanisms for several aspects of tube formation.